Several types of side-chain analogs of methotrexate (MTX) have been synthesized and are being studied in this laboratory as part of an ongoing program directed toward the development of new antifolates with favorably altered biochemical, pharmacological, and therapeutic properties. Included in this program are (a) lipid-soluble alkyl and aralkyl esters of MTX, whose ability to enter cells by passive diffusion offers an attractive approach to the treatment of MTX-resistant tumors with a transport defect, and whose nonpolar character may facilitate uptake into the central nervous system; (b) N-aryl and N-aralkyl amide "prodrugs" of MTX designed to undergo regionally selective biotransformation to the parent acid in specific catabolically active organs such as the liver, or in the vicinity of large necrotic tumors which are known to release large amounts of cathepsins and other amidases; and (c) analogs in which the glutamate moiety has been replaced by other amino acids with a terminal groups (e.g. NH2 or SH) that can be further modified to generate molecules with potentially important biological applications such as fluorescent affinity labeling and active site directed irreversible inactivation of the target enzyme dihydrofolate reductase (DHFR). Test systems available for the evaluation of new compounds generated during this program include spectrophotometric and competitive ligand-binding assays of DHFR activity; measurement of radioactive nucleoside incorporation into DNA and RNA, as well as into the acid-soluble nucleotide pool; cytotoxicity assays against cells in culture; antitumor assays in experimental animals; and distribution, biotransformation, and pharmacokinetic studies in mice and Rhesus monkeys. Ultimately we hope to identify a compound from this program whose biochemical and pharmacologic properties make it a logical candidate for clinical use.